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Updated: Jun 5, 2025
LDL Cholesterol Uptake Assay Using Live Cell Imaging Analysis with Cell Health Monitoring
Published on: November 17, 2018
MiR-99a-5p up-regulates LDLR and functionally enhances LDL-C uptake via suppressing PCSK9 expression in human
Xuemei Chen1, Ying Liu1, Qiujing Zhou1
1Department of Cell and Molecular Biology, School of Life Science and Technology, State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Druggability of Biopharmaceuticals, China Pharmaceutical University, Nanjing, Jiangsu, China.
Background:
MicroRNAs (miRs/miRNAs) play pivotal roles in modulating cholesterol homeostasis. Proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to low-density lipoprotein receptor (LDLR) at the surface of hepatocytes and accelerates its degradation in lysosomes, thereby impairing the clearance of circulating low-density lipoprotein cholesterol (LDL-C) from plasma. Thus, suppressing PCSK9 expression level has become an effective approach for treating hypercholesterolemia. Here, we sought to identify novel miRNAs that inhibit PCSK9 expression.
Methods:
By in silico analyses, miR-99a-5p was predicted to bind to human PCSK9 mRNA. Following transfection of miR-99a-5p or anti-miR-99a-5p in human and mouse hepatocytes, qRT-PCR, Western blot, immunofluorescence, ELISA, flow cytometry, LDL-C uptake, and cellular cholesterol measurement were performed.
Results:
miR-99a-5p overexpression potently inhibited PCSK9 expression, thereby up-regulating LDLR, functionally enhancing LDL-C uptake and increasing intracellular cholesterol levels in human, but not in mouse, cells. Conversely, anti-miR-99a-5p upregulates PCSK9, leading to a reduction in LDLR, attenuation of LDL-C uptake, and a decrease in the intracellular cholesterol levels of human hepatocytes. Furthermore, miR-99a-5p was shown to bind to the predicted target site "UACGGGU" in the 3'-UTR of human PCSK9 mRNA via a luciferase reporter assay in combination with site-directed mutagenesis.
Conclusion:
MiR-99a-5p potently downregulates the expression of PCSK9 by directly interacting with a target site in the human PCSK9 3'-UTR, thereby up-regulating LDLR and functionally enhancing LDL-C uptake in human hepatocytes. MiR-99a-5p could serve as an inhibitor of PCSK9 for treating hypercholesterolemia to inhibit atherosclerosis.
Insights
MicroRNA-99a-5p inhibits proprotein convertase subtilisin/kexin type 9 (PCSK9) expression, upregulating the low-density lipoprotein receptor (LDLR) and enhancing cholesterol clearance in human cells. This finding offers a potential therapeutic strategy for hypercholesterolemia.
Area of Science:
- Biochemistry
- Molecular Biology
- Genetics
Background:
- MicroRNAs (miRNAs) are key regulators of cholesterol homeostasis.
- Proprotein convertase subtilisin/kexin type 9 (PCSK9) degrades the low-density lipoprotein receptor (LDLR), impairing LDL-C clearance.
- Inhibiting PCSK9 is a validated strategy for managing hypercholesterolemia.
Purpose of the Study:
- To identify novel miRNAs that can suppress PCSK9 expression.
- To investigate the role of miR-99a-5p in regulating PCSK9 and cholesterol metabolism.
Main Methods:
- In silico prediction of miRNA targets.
- Transfection of miRNA mimics and inhibitors in human and mouse hepatocytes.
- Quantitative real-time PCR (qRT-PCR) and Western blotting to assess gene and protein expression.
- Luciferase reporter assays to confirm direct binding of miRNA to target mRNA.
Main Results:
- Overexpression of miR-99a-5p significantly inhibited PCSK9 expression in human hepatocytes.
- miR-99a-5p upregulation led to increased LDLR levels, enhanced LDL-C uptake, and elevated intracellular cholesterol.
- miR-99a-5p directly binds to the 3'-UTR of human PCSK9 mRNA.
- These effects were observed in human but not mouse hepatocytes.
Conclusions:
- MiR-99a-5p directly downregulates PCSK9 expression by binding to its 3'-UTR.
- This mechanism enhances LDLR function and promotes LDL-C uptake in human cells.
- miR-99a-5p represents a potential therapeutic agent for hypercholesterolemia and atherosclerosis.
